Membrane microdomains ("rafts") play critical roles in a number of important biological functions and pathological processes. The goal of this proposal is to investigate some of the fundamental open questions concerning raft properties and mechanisms of raft formation. Our central hypothesis is that bilayer structural and elastic properties, as controlled by hydrocarbon chain composition and cholesterol content, are critical in the sorting of both lipids and proteins between raft and non-raft membranes, as well as in regulating the function of raft channels. To study the physical-chemical, structural, and permeability properties of relevant lipid and lipid-protein systems, this application proposes to use a battery of biophysical and biochemical techniques, including confocal microscopy, interdomain partition coefficient measurements, direct measurements of raft mechanical properties by a novel application of pipette aspiration, freeze-fracture electron microscopy, X-ray diffraction, and voltage clamp experiments. This approach should yield direct and detailed information on the roles of interactions between specific raft molecules. Specific aims include determining the mechanisms involved in the sorting between raft and non-raft bilayers of phospholipids involved in signal transduction, peptides of different transbilayer lengths, and membrane channel proteins. To test models of the molecular mechanisms involved in sorting between bilayer microdomains, the distribution of lipids and peptides in bilayer microdomains will be measured, and bilayer structure and elasticity will be determined for vesicles containing: (1) both raft and non-raft domains, (2) only raft bilayers, and (3) only non-raft bilayers. In addition, as a direct functional test of rafts, the permeability and structure of these three bilayer systems will determined in the presence and absence of two raft-associated membrane channels, the connexon of gap junctions and the water channel aquaporin-0. These data will provide insights concerning the roles of lipid-lipid, lipid-protein, and protein-protein interactions in the sorting, aggregation, and permeability of these channels.